4 ms and 12.5 ms, respectively, with 71% of correlations peaking within 20 ms and 93% within 50 ms (Figure 1E). Distributions of lag times did not differ significantly between inhibitory and excitatory connections (p = 0.24, Student’s t test). Taking into account delays due to action potential propagation (0.1 to 6.5 ms; Figure S3) and postsynaptic response (15.4 ms as measured by Itri et al. [2004]), these results

suggest that a majority of the deduced connections represent direct, fast synaptic interactions and a minority (i.e., those with longer lag times) may arise from polysynaptic interactions, common inputs, or postinhibitory rebound. Because most, if not all, SCN neurons are GABAergic and express GABAA receptors, we tested whether the mapped interactions depend on GABA-mediated signaling using gabazine (Gbz, 100 μM) or bicuculline (200 μM). These Adriamycin concentration GABAA receptor antagonists decreased the number of significant connections

by 90% ± 2% (mean ± SEM) compared to vehicle (p = 0.03; Figure 1F; comparing connections from 120 randomly selected GSK1349572 price neurons in three cultures during vehicle and drug application). This loss in functional connections was not due to a decrease in firing since discharge rates actually increased slightly under GABA blockade (vehicle = 4.06 ± 0.31 Hz; mean ± SEM; GABAA-R antagonists = 5.41 ± 0.38 Hz; p < 0.05). Those few cross-correlations that remained had low Z-scores, suggesting that they could have been weakened by the blockers or could depend on an alternate, weak intercellular signal. Given the 96% hit rate of BSAC, we conclude that at least 93% of all detected interactions in these SCN cultures, both inhibitory and excitatory, depend on GABAA receptor signaling. Specific network topologies have been postulated to underlie coordinated activity in a variety of biological systems including neural networks (Grinstein and Linsker, 2005; Harris et al., 2003; Bonifazi et al., 2009). These

topologies are considered scale-free if their degree distributions follow a power law. To assess the architecture of the GABA-dependent network Amisulpride in the SCN, we measured the number of connections from and to each neuron (out and in node degrees, respectively). As indicated by spatial network maps (e.g., Figures 1D and S2), levels of single-cell connectivity were heterogeneous. Neurons received connections from a median of 4.4% of the network and sent connections to 4.5% of the network; strikingly, some cells (15 of 330 cells) were directly connected to greater than 25% of the whole population and only 1.8% of nodes remained unconnected. Out and in degree distributions did not differ significantly across cultures (p > 0.05 respectively, one-way ANOVAs) and were fit better by first-order exponential decay functions (r2 = 0.95 and 0.87, respectively) than power functions (out: r2 = 0.82, F(1,20) = 51.83, p < 0.0001; in: r2 = 0.81, F(1,15) = 8.13, p = 0.012, extra-sum-of-squares F test).